Potential of Circulating miRNA Biomarkers and Exosomes for Early Pregnancy Diagnoses in Cattle.

Circulating microRNAs (miRNAs) were investigated as biomarkers for the diagnosis of early pregnancy in cattle. The levels of prospective miRNA biomarkers and the features of extracellular vesicles (EVs) in the blood were evaluated. In Study 1, plasma samples from cows 21 days after artificial insemination (AI) were examined using RT-qPCR to determine the levels of seven circulating miRNAs. Only the levels of miR-126-3p were significantly lower in the pregnant group than in the non-pregnant group. In Study 2, among individuals not pregnant at the first AI, the miRNA levels were compared between the individuals pregnant at the second AI and those who remained non-pregnant. The miR-25 levels were significantly higher in the pregnant group at the second AI than in the pregnant group at the first AI; miR-19b, miR-27b, and miR-29a levels were also high. In the non-pregnant group, changes were absent in the miRNA levels in the same individual between the first and second AIs. In Study 3, Western blotting and RT-qPCR showed the presence of miRNAs in EVs and their levels were lower than in plasma. Thus, circulating miR-126-3p may serve as a biomarker for the diagnosis of early pregnancy in cattle. In addition, the expression of some miRNAs tended to be higher during pregnancy than during non-pregnancy in the same individual, suggesting their potential as an index to determine pregnancy and non-pregnancy rates using a comparative method.

Identification of Potential miRNA Biomarkers to Detect Hydrocortisone Administration in Horses.

Circulating microRNAs (miRNAs) are stable in bodily fluids and are potential biomarkers of various diseases and physiological states. Although several studies have been conducted on humans to detect drug doping by miRNAs, research on drugs and miRNAs in horses is limited. In this study, circulating miRNAs in horses after hydrocortisone administration were profiled and variations in miRNAs affected by hydrocortisone administration during endogenous hydrocortisone elevation were examined. The miRNAs were extracted from thoroughbred horse plasma before and after hydrocortisone administration and subjected to small RNA sequencing and reverse transcription quantitative PCR (RT-qPCR). RT-qPCR validation was performed for the 20 miRNAs that were most affected by hydrocortisone administration. The effects of elevated endogenous hydrocortisone levels due to exercise and adrenocorticotropic hormone administration were also confirmed. The validation results showed that approximately half of the miRNAs showed the same significant differences as those obtained using small RNA sequencing. Among the twenty miRNAs, two novel miRNAs and miR-133a were found to vary differently between exogenous hydrocortisone administration and endogenous hydrocortisone elevation. This study provides basic knowledge regarding the circulating miRNA profile of horses after hydrocortisone administration and identifies three miRNAs that could potentially be used as biomarkers to detect hydrocortisone administration.

Interferon-Stimulated Gene Expression in Peripheral Blood Leucocytes as a Convenient Prediction Marker for Embryo Status in Embryo-Transferred Japanese Black Cows during the Peri-Implantation Period.

Pregnancy diagnosis during early gestation is important for cattle reproduction. The expression of interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) was studied in embryo-transferred (ET) Japanese Black cattle. ISGs in PBLs-ISG15, MX1, MX2, and OAS1-were detected in multiple ovulation ET cattle using a real-time quantitative polymerase chain reaction, and receiver operating characteristic (ROC) curve analysis was performed. Gestational status was predicted using the average ISG levels during the normal estrous cycle (AVE) and the Youden index from the ROC curve analysis as cutoff values. The ISG15, MX1, and MX2 levels were significantly higher in pregnant cattle (n = 10) than in non-pregnant cattle (n = 23) on gestation day 21, whereas the levels of all ISGs were similar between non-pregnant and non-pregnant cattle with late embryonic death (n = 7). ISG15, MX1, and MX2 appropriately predicted the gestational status of ET cows. The statistical evaluation of the diagnostic accuracy in ET cows on day 21 of gestation presented higher values of sensitivity, specificity, accuracy, and positive predictive values of ISG15, MX1, and MX2 using the Youden index than using the AVE. Therefore, ISG15, MX1, and MX2 are excellent biomarkers of gestational status during the peri-implantation period in ET cattle.

A Highly Conserved Region in BRCA2 Suppresses the RAD51-Interaction Activity of BRC Repeats.

Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.

Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2.

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.

Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle.

A prediction method for early pregnancy status (pregnant or non-pregnant) in cattle that can be used within 3 weeks after insemination is desired. Interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) have been examined as prediction molecules for determination of pregnancy status. Relative abundances of ISG15 and MX2 gene transcripts in PBLs were suitable biomarkers for the prediction of pregnancy status when there were assessments of Holstein cattle. In the present study, it was determined whether ISG biomarkers are applicable for predicting gestation in Japanese-Black (JB) cattle and evaluation of the applicability of receiver operating characteristic (ROC) analysis procedures for this purpose. There was assessment of the reliability of using average ISG values in PBLs collected during the estrous cycle (AVE) as a cutoff compared to the Youden index cutoff values. Application of AVE to assessment of pregnancy status in JB cattle indicated there was reliable predictions for pregnancy status when using ISG15 and MX2 values on day 21 after insemination, which coincided with the time of assessment in the previous study with Holstein cattle. The area under the curve values of the ROC curves confirmed the reliability of using ISGs to predict pregnancy from days 18 to 21 after insemination. Comparing AVE with Youden index values, there was confirmation of the accuracy of AVE for predicting gestation. The average mRNA transcript abundance values of ISG15 and MX2 may serve as excellent pregnancy biomarkers for cattle within 3 weeks of insemination.

Gene expression profiles in bovine granulocytes reflect the aberration of liver functions.

Liver performs several important functions; however, predicting its functions is difficult. Methods of analyzing gene expression profiles, for example, microarray, provide functional information of tissues. Liver and peripheral blood leukocytes (PBLs) were collected from Holstein cows subjected to two different physiological conditions (non-pregnant and pregnant), and PBLs were fractionated by gradient cell separation. RNA from PBLs and liver were applied to oligo-DNA microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). It revealed a group of stable bovine liver genes under constant physiological conditions. When they applied to physiological conditions including non-pregnant and pregnant, the profiles of some genes in liver were consistent with those in PBLs. Microarray data subjected to a principal component analysis (PCA) showed that the hepatic gene expression profiles were more consistent with those of granulocytes than mononuclear cells. The relationship of gene profiles in liver with granulocytes was confirmed by RT-qPCR and hierarchical cluster analysis. Gene profiles of granulocytes were more reliable than those of mononuclear cells, which reflected liver functions. These results suggest that the genes expressed in PBLs, particularly granulocytes, may be convenient bioindicators for the diagnosis of clinical disorder and/or detecting aberration of liver functions in cows subjected to different physiological conditions.

A cell-based interferon-tau assay with an interferon-stimulated gene 15 promoter.

Interferon-tau (IFNT) is known as an early pregnancy recognition signal in ruminants. An accurate and convenient IFNT detection system is desirable for the diagnosis of endometrial and trophoblastic functions, including gestation status, in cows. The aim of this study was to develop a new cell-based assay, which involved the stable introduction of an interferon-stimulated gene promoter to a luciferase reporter system. The reactivity of four interferon-stimulated genes to IFNT in Madin-Darby bovine kidney (MDBK) cells was confirmed using reverse transcription-quantitative PCR. The upstream region of the interferon-stimulated gene 15 ubiquitin-like modifier (ISG15) gene as the promoter of the reporter gene, which is more responsive to IFNT and other IFNs, was determined using the luciferase assay. The reporter gene with the ISG15 upstream region was stably transfected into MDBK cells using the PiggyBac vector system; this cell line responded to type I IFNs in a dose-dependent manner. Because of its convenience, this cell line is suitable for the quantification of IFNT as well as other type I IFNs activities.

R132 mutations in canine isocitrate dehydrogenase 1 (IDH1) lead to functional changes.

Glioma is the second most common intracranial neoplasia in dogs, but the pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas. Although almost all human IDH1 mutations have been identified as involving the Arg132 codon, few studies have reported structural, functional, and mutational information for canine IDH1. Therefore, in this study, we cloned the canine IDH1 homologue and used PCR mutagenesis to substitute the wildtype (WT) Arg132 with His (R132H) or Ser (R132S). WT and mutated IDH1 were overexpressed in HeLa cells, and their presence was confirmed by immunoblotting and immunocytochemistry using mutation-specific antibodies. The IDH1 activity between WT, R132H, and R132S transfectants was compared by measuring the production of NADH and NADPH. NADPH production in R132H and R132S transfectants was lower than that in WT, but NADH levels were not significantly different. Finally, we detected increased expression of hypoxia inducible factor 1 alpha (HIF-1α) in the R132H and R132S transfectants. These results indicated that the canine IDH1 Arg132 mutation has the potential to induce carcinogenesis in canine somatic cells.

A predictive threshold value for the diagnosis of early pregnancy in cows using interferon-stimulated genes in granulocytes.

Interferon tau plays an important role in establishing bovine pregnancy. Interferon-stimulated genes (ISGs) have been examined to identify a suitable indicator for the diagnosis of early gestation in cows. Although ISGs can be specifically detected in peripheral white blood cells during early gestation, its reliability remains to be validated. In the current study, a predictive threshold level of ISGs to determine pregnancy in cows during Days 20-22 of gestation was verified by analyzing the expression of ISGs in granulocytes and peripheral blood leucocytes (a total of 57 cows were used, 28 of which were pregnant and 29 were non-pregnant). Four genes, interferon-stimulated gene 15 ubiquitin-like modifier (ISG15), MX dynamin like GTPase (MX) 1, MX2, and 2'-5'-oligoadenylate synthetase 1 (OAS1), were analyzed via quantitative RT-PCR and a receiver operating characteristic (ROC) curve was produced to visualize diagnostic accuracy measures. The expression values of the four ISGs during the estrous cycle (100 collection points from 65 cattle) were used to determine a pregnancy prediction cutoff value. Pregnancy status was determined using these cutoff values and then confirmed by ultrasonography. ROC analysis was then applied to confirm the accuracy of the pregnancy statuses (positive and negative) statistically. The statistical evaluation of the diagnostic accuracy measurements suggested that the average values of ISG15 and MX2 in granulocytes were reliable indicators of pregnancy within the three weeks after insemination with 80% accuracy. Average ISG15 and MX2 levels during the estrous cycle were more reliable biomarkers for the prediction of gestation. They predicted negative and positive pregnancies efficiently within three weeks after artificial insemination.

Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRß antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity.

REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling.

Aberrant gene expression of heparanase in ventricular hypertrophy induced by monocrotaline in rats.

The gene expression levels of heparanase, matrix metalloproteinase 2 (MMP2) and MMP9 were examined in ventricles after treatment with monocrotaline (MCT) to induce cardiac hypertrophy in rats. Rats received a single intraperitoneal injection of MCT (60 mg/kg) or saline. Twenty-five days after the injection, the right ventricle and lung wet weights were increased in MCT-treated rats compared with the control. Histological analysis revealed cardiomyocyte hypertrophy in the right ventricle of MCT-treated rats. Northern blot hybridization showed that heparanase and MMP2 expression increased significantly in the right and left ventricles of MCT-treated rats, whereas MMP9 was not induced. These findings indicate that heparanase and MMP2 might play an important role in the development of MCT-induced cardiac hypertrophy.

Induction of ovine trophoblast cell fusion by fematrin-1 in vitro.

Endogenous retroviruses present in the genomes take a specific role in placental formation in various vertebrates, including bovine and sheep. Fematrin-1, which is the envelope (Env) protein of bovine endogenous retrovirus found in bovine placenta, is involved in the formation of fetomaternal hybrid cells in cattle placenta. This study was conducted to clarify whether fematrin-1 possesses fusogenic activity in trophoblast cells. Another question is whether Env proteins only have species-specific activity or not. For this, fematrin-1 gene was transfected in ovine trophoblast cells, and we examined fusogenic activity with Cos-7 cells. Although fematrin-1 fusogenic activity was detected in both neutral and acidic pH conditions, acidic condition significantly enhanced it. These activities were rather weaker than those of vesicular stomatitis virus G protein as a positive control. However, the ratio of fematrin-1 and vesicular stomatitis virus G protein fusion index was confirmed similar to those in the previous reports. Some fusion cells showed multinucleate cells. These results imply that fematrin-1 is involved in the formation of trophoblast hybrid cells even in different species trophoblastic cells.

Nfkbiz regulates the proliferation and differentiation of keratinocytes.

Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) inhibitor zeta (Nfkbiz) is a nuclear inhibitor of NF-κB (IκB) protein that is also termed as molecule possessing ankyrin repeats induced by lipopolysaccharide, interleukin-1-inducible nuclear ankyrin repeat protein, or IκBζ. We found previously that disrupting the Nfkbiz gene resulted in atopic dermatitis-like lesions in mice, suggesting an important role for Nfkbiz in the skin. In this study, we examined the cellular function of Nfkbiz in keratinocytes. Immunohistochemical analyses for Ki-67 revealed that Nfkbiz-/- keratinocytes were hypoproliferative. In skin from Nfkbiz-/- mice, the expression of the keratinocyte differentiation markers K10 and filaggrin were reduced, although that of K14 was unchanged. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that the frequency of apoptosis was comparable between control and Nfkbiz-/- keratinocytes. Interestingly, the subcellular localization of the NF-κB subunits and the transcriptional activity of NF-κB were not changed in Nfkbiz-/- keratinocytes. These findings indicate a novel possible role of Nfkbiz in controlling the proliferation and differentiation of epidermal keratinocytes through NF-κB-independent mechanisms.

Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer.

Although the morbidity of canine prostate cancer is low, the majority of cases present with resistance to androgen therapy and poor clinical outcomes. These pathological conditions are similar to the signs of the terminal stage of human androgen-independent prostate cancer. The co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) is known to be overexpressed in human androgen-independent prostate cancer. However, there is little information about the structure and function of canine SGTA. In this study, canine SGTA was cloned and analysed for its ability to suppress androgen receptor signalling. The full-length open reading frame (ORF) of the canine SGTA gene was amplified by RT-PCR using primers designed from canine-expressed sequence tags that were homologous to human SGTA. The canine SGTA ORF has high homology with the corresponding human (89%) and mouse (81%) sequences. SGTA dimerisation region and tetratricopeptide repeat (TPR) domains are conserved across the three species. The ability of canine SGTA to undergo homodimerisation was demonstrated by a mammalian two-hybrid system and a pull-down assay. The negative impact of canine SGTA on androgen receptor (AR) signalling was demonstrated using a reporter assay in androgen-independent human prostate cancer cell lines. Pathological analysis showed overexpression of SGTA in canine prostate cancer, but not in hyperplasia. A reporter assay in prostate cells demonstrated suppression of AR signalling by canine SGTA. Altogether, these results suggest that canine SGTA may play an important role in the acquisition of androgen independence by canine prostate cancer cells.

Immunohistochemical localization of GLUT3, MCT1, and MCT2 in the testes of mice and rats: the use of different energy sources in spermatogenesis.

Lactate represents a preferential energy substrate of germ cells rather than glucose. Testicular Sertoli cells are believed to produce lactate and pyruvate and to supply these to germ cells, particularly spermatocytes and spermatids. Monocarboxylate transporter (MCT), responsible for the transport of lactate and other monocarboxylates via the cell membrane, is abundant in the testes and sperm (MCT1, MCT2, and MCT4). For the uptake of glucose, germ cells within the seminiferous tubules and sperm have been known to intensely express GLUT3. The present study investigated expression profiles of MCTs and GLUTs and revealed their cellular and subcellular localization in the mouse and rat testis. An in situ hybridization analysis showed significant expressions of MCT1, MCT2, and GLUT3 mRNA in the testis. Immunohistochemically, spermatogonia, spermatocytes, and spermatids expressed MCT1 on their cell surfaces in a stage-dependent manner: in some seminiferous tubules, an intense expression of MCT1 was unique to the spermatogonia. MCT2 was restricted to the tails of elongated spermatids and sperm. An intense immunoreactivity for GLUT3 was shared by spermatocytes, spermatids, and sperm. Sertoli cells were devoid of any immunoreactivities for MCT1, MCT2, and GLUT3. The predominant energy source of germ cells may be lactate and other monocarboxylates--especially for spermatogonia, but glucose and other hexoses may be responsible for an energy supply to spermatocytes and spermatids.

Reduced canine BRCA2 expression levels in mammary gland tumors.

BACKGROUND: Mammary tumors are the most common tumor type in intact female dogs. Recently, the breast cancer 2 early onset (BRCA2) gene was proposed to be associated with tumorigenesis in dogs. The expression level of BRCA2 is important for its DNA repair function in mammalian cells, and its expression level is linked to tumorigenesis in mammary tissue. However, the expression of canine BRCA2 in mammary tumors is unclear. RESULTS: BRCA2 mRNA levels were compared between seven mammary gland samples and seventeen mammary tumor samples isolated from dogs. The expression level of canine BRCA2 in mammary tumor samples was lower than levels in mammary gland samples. We attempted to identify why the BRCA2 expression level was decreased in mammary tumor samples by promoter sequencing analysis; however, we did not find any mutations in the canine BRCA2 promoter that altered BRCA2 transcription levels. We did detect two types of BRCA2 splice variants in 8 mammary tumor samples. One of the variants induced a frame-shift mutation that could lead to nonsense-mediated mRNA decay, a ubiquitous cellular mechanism that eliminates mRNA containing a premature termination codon. CONCLUSIONS: Reduced expression of canine BRCA2 mRNA in mammary tumor samples is a possible mechanism to explain mammary tumor development in dogs. One possible reason for reduced BRCA2 mRNA levels in these tumor samples was nonsense-mediated mRNA decay, not mutations in the BRCA2 promoter region. While it remains unclear why canine BRCA2 expression levels are reduced in mammary tumor samples, this study found that the expression level of BRCA2 was associated with canine mammary tumorigenesis.

The role of IFN-γ in regulating Nfkbiz expression in epidermal keratinocytes.

Nfkbiz is an inhibitor of nuclear factor κB (IκB) protein localized to the nucleus. We previously found that Nfkbiz gene-disrupted mice showed atopic dermatitis-like lesion, implying the important role of Nfkbiz in skin homeostasis. The purpose of this study was to examine the effect of interferon (IFN)-γ on Nfkbiz expression in keratinocytes. IFN-γ induced Nfkbiz expression at a comparable level to IL-1. Promoter analysis revealed that interferon-stimulated response element (ISRE) located in the Nfkbiz promoter region is important for responding to the stimulation. Interestingly, IFN-γ and IL-1 displayed synergism in terms of inducing Nfkbiz expression. By using selective inhibitors, we found that Janus activated kinase (JAK) 1 and nuclear factor (NF)-κB are important for Nfkbiz expression after IFN-γ stimulation and for synergism between IFN-γ and IL-1. These findings indicate a possible important role of Nfkbiz in modulating the progression of inflammatory diseases in which IFN-γ and IL-1 are abundant.